<p>Waste tire rubber (WTR) has been introduced as an alternative, novel media for biofilm development in several experimental systems including attached growth bioreactors. In this context, four laboratory-scale static batch bioreactors containing WTR as a support material for biofilm development were run under anoxic condition for 90 days using waste activated sludge as an inoculum under the influence of different concentrations (2.5, 6.5, 8.5 mg/l) of trivalent ferric iron (Fe<sup>3+</sup>). The data revealed that activated sludge with a Fe<sup>3+</sup> concentration of 8.5 mg/l supported the maximum bacterial biomass [4.73E + 10 CFU/ml cm<sup>2</sup>]; besides, it removed 38% more Chemical oxygen demand compared to Fe<sup>3+</sup> free condition from the reactor. Biochemical testing and 16S rDNA phylogenetic analysis of WTR-derived biofilm communities further suggested the role of varying concentrations of Fe<sup>3+</sup> on the density and diversity of members of Enterobacteria(ceae), ammonium (AOB) and nitrite oxidizing bacteria. Furthermore, Fluorescent <i>in situ</i> hybridization with phylogenetic oligonucleotide probes and confocal laser scanning microscopy of WTR biofilms indicated a significant increase in density of eubacteria (3.00E + 01 to.05E + 02 cells/cm<sup>2</sup>) and beta proteobacteria (8.10E + 01 to 1.42E + 02 cells/cm<sup>2</sup>), respectively, with an increase in Fe<sup>3+</sup> concentration in the reactors, whereas, the cell density of gamma proteobacteria in biofilms decreased.</p>